To cause disease, pathogenic bacteria must overcome host innate immune barriers to infection. Interferon gamma (IFNy) and tumor necrosis factor alpha (TNFa) are cytokines produced both early and later in the immune response. Early production of these cytokines rapidly activates inflammatory responses to serve as a first line of defense against invading bacteria. We and other labs have found that Francisella tularensis and Burkholderia pseudomallei interfere with NFicB activation (causing reduced production of TNFa) and with the response of infected cells to IFNy. We hypothesize that subversion of these defense pathways involves specific pathogen gene products ("effectors") and promotes infection and disease caused by these category A and B cytosolic bacterial pathogens. We have developed novel screening technologies to identify the Francisella tularensis and Burkholderia pseudomallei gene products responsible for their subversion of host IFNy and NFicB responses. Effector proteins identified in our screens will be studied for their contributions to immune subversion and disease in infected animals. We will also dissect their mechanisms of action and develop assays that will facilitate screening for inhibitors of their functions. These studies will thus lay the foundation for future work to therapeutically impair establishment of F. tularensis and B. pseudomallei infections. Our Specific Aims are: (1) Identify F. tularensis and B. pseudomallei gene products interfering with innate immune responses. (2) Engineer mutant F. tularensis and B. pseudomallei strains deficient for putative immune-suppressive genes and test the effects of such mutations on virulence in mouse infection models. (3) Identify the stage in the macrophage response to IFNy that is impaired by infection with F. tularensis. We will use novel reagents and new technologies to identify novel therapeutic targets and attenuated vaccine strains in F. tularensis and B. pseudomallei. Further, by demonstrating how these technologies can be applied, our studies may ultimately promote the identification and therapeutic targeting of immune modulating virulence proteins from other select agents and emerging pathogens. This research project fits within the RMRCE Integrated Research Focus on Bacterial Therapeutics, will interact directly with RP(s) 1.4, 1.5, 2.1, 2.5, and 2.6, and will utilize the resources of Core(s) C, E, and F.